LC-MS-MS

基于货车Breemen博士实验室发明的脉冲超滤质谱法，将快速筛选天然产物提取物，以发现与重要靶分子结合的特定化合物。这些高通量筛选试验将提高整个项目的生产率。将利用现有的用于环氧合酶-2和雌激素受体-α和-β的配体的超滤筛选测定，以及将开发的用于筛选受体如雄激素受体和RXR的配体的新测定。 超滤质谱筛选期间获得的命中将基于分子量、元素组成（使用精确质量测量确定）、串联质谱（MS-MS）裂解模式、与UIC合作研究计划中可用的标准品的比较， 药物科学，使用UIC NAPRALERT天然产物数据库和HPLC纯化材料的质子NMR和13 C-NMR分析进行去复制。由于这种筛选方法将基于与特定靶蛋白的结合来鉴定先导化合物，因此这些化合物可以用于药物筛选。 将在核心B中测定命中物以确认其生物活性。接下来，将通过与人肝细胞和人肝微粒体孵育，然后使用LC-MS和LC-MS-MS进行分析，在体外评估来自该计划所有部分的先导化合物的I期和II期代谢。将通过LC-MS和LC-MS-MS分析来自动物研究的血液和尿液样品，评价癌症化学预防剂的体内代谢。此外，将在动物中进行这些化合物的药代动力学研究。在另一组临床前药物开发研究中，将使用Caco-2细胞单层模型评价每种先导化合物的人体肠道渗透性。最后，将使用LC-MS和LC-MS-MS确认化合物的分子量、元素组成、裂解模式和纯度。总之，本项目将通过筛选天然产物提取物以快速鉴定特定靶蛋白的配体，通过提供先导化合物的肝脏代谢、药代动力学和肠道渗透性的临床前评价，以及通过提供分离和合成化合物的质谱表征，与本项目中的所有其他项目相互作用。

Based on the method of pulsed ultrafiltration mass spectrometry, which was invented in the laboratory of Dr. van Breemen, natural product extracts will be screened rapidly for the discovery of specific compounds that bind to important target molecules. These high throughput screening assays will enhance the productivity of the entire program. Existing ultrafiltration screening assays for ligands to cyclooxygenase-2 and the estrogen receptors-alpha and -beta will be utilized as well as new assays that will be developed to screen for ligands to receptors such as the androgen receptor and RXR. Hits obtained during ultrafiltration mass spectrometric screening will be identified based on molecular weight, elemental composition (determined using exact mass measurement), tandem mass spectrometric (MS-MS) fragmentation patterns, comparison with standards available at the UIC Program for Collaborative Research in the Pharmaceutical Sciences, dereplication using the UIC NAPRALERT database of natural products and proton NMR and 13C-NMR analysis of HPLC-purified material. Since this screening approach will identify lead compounds based on binding to a specific target protein, these hits will be assayed in Core B to confirm their biological activity. Next, the Phase I and Phase II metabolism of the lead compounds from all parts of the program will be assessed in vitro by incubation with human hepatocytes and human liver microsomes followed by analysis using LC-MS and LC-MS-MS. The in vivo metabolism of cancer chemoprevention agents will be evaluated by LC-MS and LC-MS-MS analysis of blood and urine samples from animal studies. In addition, pharmacokinetic studies of these compounds will be carried out in animals. In another set of preclinical drug development studies, the human intestinal permeability of each lead compound will be evaluated using the Caco-2 cell monolayer model. Finally, LC-MS and LC-MS-MS will be used to confirm the molecular weights, elemental compositions, fragmentation patterns, and purity of compounds. In summary, this project will interact with all of the other Projects within this program by screening natural product extracts for the rapid identification of ligands for specific target proteins, by providing preclinical evaluation of hepatic metabolism, pharmacokinetics and intestinal permeability of lead compounds, and by providing mass spectrometric characterization of isolated and synthetic compounds.